This invention pertains to the fields of immunodiagnostics and immunotherapeutics. The invention provides methods of identifying internalizing antibodies and internalizing receptor ligands, as well as the internalizing receptors bound.
Growth factor receptors, and other signal transduction receptors, are frequently overexpressed in human carcinomas and other diseases and thus have been utilized for the development of targeted therapeutics. The HER2/neu gene, for example, is amplified in several types of human adenocarcinomas, especially in tumors of the breast and the ovary (Slamon et al. (1989) Science 244: 707-712) leading to the overexpression of the corresponding growth factor receptor ErbB2. Targeting of ErbB2 overexpressing cells has been accomplished primarily using anti-ErbB2 antibodies in different formats, including conjugation to liposomes containing chemotherapeutics (Kirpotin et al. (1997). Biochem. 36: 66-75), fusion to DNA carrier proteins delivering a toxic gene (Forminaya and Wels (1996) J. Biol Chem. 271: 10560-10568), and direct fusion to a toxin (Altenschmidt et al. (1997) Int. J Cancer 73: 117-124).
For many of these targeted approaches, it is necessary to deliver the effector molecule across the cell membrane and into the cytosol. In some cases, this can be facilitated by taking advantage of receptor mediated endocytosis (Ullrich and Schlessinger (1990) Cell 61: 203-212). Receptor-mediated endocytosis is often caused when ligand binding causes receptor activation via homo- or heterodimerization, either directly for bivalent ligand or by causing a conformational change in the receptor for monovalent ligand. Antibodies can mimic this process, stimulate endocytosis, become internalized and deliver their payload into the cytosol. In addition, the efficiency with which antibodies mediate internalization differs significantly depending on the type of the antibody (e.g. whole antibody, fragment, single chain, monomeric, dimeric, etc.) and on the epitope recognized (Yarden (1990) Proc. Natl. Acad. Sci. USA 87: 2569-2573; Hurwitz et al (1995) Proc. Natl. Acad. Sci. USA 92: 3353-3357.). Thus for some applications, such as liposomal targeting, only antibodies that bind specific epitopes are rapidly internalized and yield a functional targeting vehicle.
Internalizing antibodies have also been shown to cause cell growth inhibition or enhanced cell growth, depending on the epitope recognized. Thus selection for internalization should lead to the isolation of growth inhibitory or stimulatory (agonist) antibodies. Such inhibitory antibodies might be used as cancer treatments or for the treatment of other conditions characterized by cell hyperproliferation, and for the treatment of inflammation (anti-inflammatories). Agonist antibodies could be used for stimulating growth of relevant cells (for example stem cells). Targeting of cells besides cancer cells for gene delivery will also have many application
Currently, antibodies that mediate internalization are identified by screening hybridomas. Screening of hybridoma-produced antibodies, however, is laborious, time-consuming, and expensive.
This invention is based, in part, on the discovery that it is possible to directly select internalizing antibodies from large non-immune phage libraries by recovering infectious phage particles from within cells after receptor mediated endocytosis.
Thus, in one embodiment, this invention provides methods of selecting polypeptide or antibody binding moieties that are internalized into target cells. The methods preferably involve i) contacting one or more of target cells with one or more members of a phage display library; iv) culturing the target cells under conditions where members of the display library can be internalized if bound to an internalizing marker; and v) identifying internalized members of the phage display library if members of the phage display library are internalized into one or more of the target cells. The methods also optionally, and preferably additionally involve contacting members of the phage display library with a cells of a subtractive cell line; and then washing the target cells to remove the cells of a subtractive cell line and to remove members of the phage display library that are non-specifically bound or weakly bound to the target cells. In a preferred embodiment, the phage display library is an antibody phage display library, more preferably an antibody phage display library displaying single chain antibodies (e.g. scFv, scFab, etc.).
In a preferred embodiment, the xe2x80x9cidentifyingxe2x80x9d step comprises recovering internalized phage and repeating steps the process again to further select for internalizing binding moieties. In one embodiment, the xe2x80x9crecoveringxe2x80x9d step involves lysing the target cells to release internalized phage; and infecting a bacterial host with the internalized phage to produce phage for a subsequent round of selection. The recovering step can involve recovering infective phage, and/or recovering a nucleic acid encoding a phage-displayed antibody and/or selection of phage expressing a selectable marker (e.g. an antibiotic resistance gene or cDNA). The identifying step can involve detecting expression of a reporter gene, detecting the presence absence or quantity of a particular nucleic acid, or selection of phage via a selectable marker. In preferred methods the cells of a subtractive cell line are present in at least 2-fold excess over the target cells. In preferred methods, the target cells form an adherent layer. In preferred methods the target cell line is grown adherent to a tissue culture plate and co-incubated with the subtracting cell line in suspension in a single cell culture flask. In particular preferred methods, the contacting with a subtractive cell line is performed at a temperature (e.g. at about 4xc2x0 C.) lower than the internalization culture conditions (e.g. at about 37xc2x0)
In particularly preferred embodiments, the phage express a selectable maker and/or a reporter gene. Preferred selectable markers include, but are not limited to genes (or cDNAs) encoding fluorescent protein(s), an antibiotic resistance gene or cDNA, and a chromagenic gene or cDNA (e.g., horse radish peroxidase, xcex2-lactamase, luciferase, and xcex2-galactosidase. In certain embodiments the target cells can include solid tumor cells, members of a cDNA expression library, cells that overexpress a cytokine receptor, cells that overexpress a growth factor receptor, metastatic cells, cells of a transformed cell line, cells transformed with a gene or cDNA encoding a specific surface target receptor, and neoplastic cells derived from outside a solid tumor. In one particularly preferred embodiment, the said cells of a subtractive cell line are selected from the same tissue type as the target cells. Suitable s subtractive cell line cells include, but are not limited to fibroblasts, monocytes, stem cells, and lymphocytes.
The methods of this invention can also be used to identify internalizing receptors and/or internalizing receptor epitopes (regions of the receptor that when bound induce internalization of the binding moiety). The methods generally involve any of the methods for identifying internalizing antibodies or polypeptides as described herein with the additional steps whereby the internalizing antibodies or polypeptides identified are used to probe the original target cells, or different cells. When the internalizing antibodies or polypeptides so bind, they permit isolation of the cell bearing the internalizing receptor and isolation of the receptor and/or receptor epitope itself. Thus in one embodiment the methods involve i) contacting one or more of the target cells with one or more members of a phage display library; ii) optionally, but preferably, contacting members of the phage display library with a cells of a subtractive cell line; iii) optionally, but preferably washing the target cells to remove said cells of a subtractive cell line and to remove members of the phage display library that are non-specifically bound or weakly bound to said target cells; iv) culturing the cells under conditions where members of said phage display library can be internalized if bound to an internalizing marker; v) identifying internalized members of the phage display library if members of the phage display library are internalized into one or more of said target cells; vi) contacting the same or different target cells with the identified internalized members of step (v) or members propagated therefrom, whereby the members bind to the surface of said same or different target cells. The method can further involve isolating a component of the same or different target cells to which the members bind. In some methods the xe2x80x9cidentifyingxe2x80x9d step involves recovering internalized phage and repeating steps (i) through (v) to further select for internalizing binding moieties.
The contacting, washing, culturing, and identifying steps are preferably performed as described herein and the target and subtractive cells include the cells described herein.
In still another embodiment, this invention provides a multivalent antibody phage display library. The library preferably comprises a plurality of phage wherein the phage display, on average, at least two copies of a single-chain antibody and the library comprises a plurality of species of single-chain antibody. In preferred embodiments, the phage display, on average, at least 3, at least 4, or at least 5 copies of a single chain antibody per phage particle. Particularly preferred libraries comprise, on average, at least 105, preferably at least 106, more preferably at least 107, and most preferably at least 108 different species of single chain antibody. In a most preferred embodiment, the antibodies are encoded by a nucleic acids that are phage (not phagemid) vectors.
In certain embodiments, the library will be selected for members that specifically bind to an internalizing cell surface receptor (e.g. erbB2, EGF receptor, PDGF receptor, VEGF receptor, transferrin receptor, etc.). The single-chain antibodies are preferably single chain Fv (scFv) or single-chain Fab (scFab) antibodies. Filamentous phage are preferably used in the libraries of this invention and the antibodies are preferably expressed as a fusion with a PIII minor coat protein. The phage can also express a selectable marker (e.g. an antibiotic resistance gene or cDNA) and/or a reporter gene or cDNA (e.g., green fluorescent protein (GFP), Fflux, xcex2-gal, xcex2-lactamase, etc.).
In still yet another embodiment, this invention provides a nucleic acid library encoding one of the phage display antibody libraries describe herein. The nucleic acid library comprises at least 105, more preferably at least 106, and most preferably at least 107 different phage or phagemid vectors.
This invention also provides kits for practice of the methods described herein. The kits preferably comprise one or more containers containing a phage display library (or a portion thereof) described herein. The kit can include nucleic acids encoding the library and/or phage particles expressing single chain antibodies (preferably a multivalent library) and/or cells containing the intact phage or nucleic acids from the phage.
As used herein, an xe2x80x9cantibodyxe2x80x9d refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fabxe2x80x2)2 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fabxe2x80x2 fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (scFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer which may be expressed from a nucleic acid including VH- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv""s (scFv), however, alternative expression strategies have also been successful. For example Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to g3p (see, e.g., U.S. Pat. No: 5,733,743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778). Particularly preferred antibodies include all those that have been displayed on phage I think preferred antibodies should include all that have been displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiter et al. (1995) Protein Eng. 8: 1323-1331).
An xe2x80x9cantigen-binding sitexe2x80x9d or xe2x80x9cbinding portionxe2x80x9d refers to the part of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (xe2x80x9cVxe2x80x9d) regions of the heavy (xe2x80x9cHxe2x80x9d) and light (xe2x80x9cLxe2x80x9d) chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as xe2x80x9chypervariable regionsxe2x80x9d which are interposed between more conserved flanking stretches known as xe2x80x9cframework regionsxe2x80x9d or xe2x80x9cFRsxe2x80x9d. Thus, the term xe2x80x9cFRxe2x80x9d refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen binding xe2x80x9csurfacexe2x80x9d. This surface mediates recognition and binding of the target antigen. The three hypervariable regions of each of the heavy and light chains are referred to as xe2x80x9ccomplementarity determining regionsxe2x80x9d or xe2x80x9cCDRsxe2x80x9d and are characterized, for example by Kabat et al. Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, Md. (1987).
As used herein, the terms xe2x80x9cimmunological bindingxe2x80x9d and xe2x80x9cimmunological binding propertiesxe2x80x9d refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the xe2x80x9con rate constantxe2x80x9d (kon) and the xe2x80x9coff rate constantxe2x80x9d (koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of koff/kon enables cancellation of all parameters not related to affinity and is thus equal to the dissociation constant Kd (see, generally, Davies et al. (1990) Ann. Rev. Biochem., 59: 439-473.
The phrase xe2x80x9cspecifically binds to a proteinxe2x80x9d or xe2x80x9cspecifically immunoreactive withxe2x80x9d, when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, F5 or C1 antibodies can be raised to the c-erbB-2 protein that bind c-erbB-2 and not to other proteins present in a tissue sample. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d, or xe2x80x9cproteinxe2x80x9d are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are preferably in the natural xe2x80x9cLxe2x80x9d isomeric form. However, residues in the xe2x80x9cDxe2x80x9d isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. In addition, the amino acids, in addition to the 20 xe2x80x9cstandardxe2x80x9d amino acids, include modified and unusual amino acids, which include, but are not limited to those listed in 37 CFR ┘1.822(b)(4). Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group.
The term xe2x80x9cbinding polypeptidexe2x80x9d refers to a polypeptide that specifically binds to a target molecule (e.g. a cell receptor) in a manner analogous to the binding of an antibody to an antigen. Binding polypeptides are distinguished from antibodies in that binding polypeptides are not ultimately derived from immunoglobulin genes or fragments of immunoglobulin genes.
The term xe2x80x9cconservative substitutionxe2x80x9d is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
The term xe2x80x9cnucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; and Cassol et al. (1992); Rossolini et al., (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The terms xe2x80x9cisolatedxe2x80x9d or xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. However, the term xe2x80x9cisolatedxe2x80x9d is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu.
A chimeric molecule is a molecule in which two or more molecules that exist separately in their native state are joined together to form a single molecule having the desired functionality of all of its constituent molecules. While the chimeric molecule may be prepared by covalently linking two molecules each synthesized separately, one of skill in the art will appreciate that where the chimeric molecule is a fusion protein, the chimera may be prepared de novo as a single xe2x80x9cjoinedxe2x80x9d molecule.
A fusion protein is a chimeric molecule in which the constituent molecules are all polypeptides and are attached (fused) to each other through terminal peptide bonds so that the chimeric molecule is a continuous single-chain polypeptide. The various constituents can be directly attached to each other or can be coupled through one or more peptide linkers.
An effector moiety or molecule is a molecule or moiety that typically has a characteristic activity that is desired to be delivered to a target cell (e.g. a tumor overexpressing c-erbB-2). Effector molecules include cytotoxins, labels, radionuclides, ligands, antibodies, drugs, liposomes, and viral coat proteins that render the virus capable of infecting a c-erbB-2 expressing cell.
A xe2x80x9ctargetxe2x80x9d cell refers to a cell or cell-type that is to be specifically bound by a member of a phage display library or a chimeric molecule of this invention. Preferred target cells are cells for which an internalizing antibody or binding polypeptide is sought. The target cell is typically characterized by the expression or overexpression of a target molecule that is characteristic of the cell type. Thus, for example, a target cell can be a cell, such as a tumor cell, that overexpresses a marker such as c-erbB-2.
A xe2x80x9ctargeting moietyxe2x80x9d refers to a moiety (e.g. a molecule) that specifically binds to the target molecule. Where the target molecule is a molecule on the surface of a cell and the targeting moiety is a component of a chimeric molecule, the targeting moiety specifically binds the chimeric molecule to the cell bearing the target. Where the targeting moiety is a polypeptide it can be referred to as a xe2x80x9ctargeting polypeptidexe2x80x9d.
The terms xe2x80x9cinternalizingxe2x80x9d or xe2x80x9cinternalizedxe2x80x9d when used in reference to a cell refer to the transport of a moiety (e.g. phage) from outside to inside a cell. The internalized moiety can be located in an intracellular compartment, e.g. a vacuole, a lysosome, the endoplasmic reticulum, the golgi apparatus, or in the cytosol of the cell itself.
An internalizing receptor or marker is a molecule present on the external cell surface that when specifically bound by an antibody or binding protein results in the internalization of that antibody or binding protein into the cell. Internalizing receptors or markers include receptors (e.g., hormone, cytokine or growth factor receptors) ligands and other cell surface markers binding to which results in internalization.
The term xe2x80x9cheterologous nucleic acidxe2x80x9d refers to a nucleic acid that is not native to the cell in which it is found or whose ultimate origin is not the cell or cell line in which the xe2x80x9cheterologous nucleic acidxe2x80x9d is currently found.
The idiotype represents the highly variable antigen-binding site of an antibody and is itself immunogenic. During the generation of an antibody-mediated immune response, an individual will develop antibodies to the antigen as well as anti-idiotype antibodies, whose immunogenic binding site (idiotype) mimics the antigen. Anti-idiotypic antibodies can also be generated by immunization with an antibody, or fragment thereof.,
A xe2x80x9cphage display libraryxe2x80x9d refers to a collection of phage (e.g., filamentous phage) wherein the phage express an external (typically heterologous) protein. The external protein is free to interact with (bind to) other moieties with which the phage are contacted. Each phage displaying an external protein is a xe2x80x9cmemberxe2x80x9d of the phage display library.
An xe2x80x9cantibody libraryxe2x80x9d refers to phage display library that displays antibodies (binding proteins encoded by one or more antibody genes or cDNAs). The antibody library includes the population of phage or a collection of vectors encoding such a population of phage, or cell(s) harboring such a collection of phage or vectors. The library can be monovalent, displaying on average one single-chain antibody per phage particle or multi-valent displaying, on average, two or more single chain antibodies per viral particle. Preferred antibody libraries comprise on average more than 106, preferably more than 107, more preferably more than 108, and most preferably more than 109 different members (i.e. encoding that many different antibodies).
The term xe2x80x9cfilamentous phagexe2x80x9d refers to a viral particle capable of displaying a heterogenous polypeptide on its surface. Although one skilled in the art will appreciate that a variety of bacteriophage may be employed in the present invention, in preferred embodiments the vector is, or is derived from, a filamentous bacteriophage, such as, for example, f1, fd, Pf1, M13, etc. The filamentous phage may contain a selectable marker such as tetracycline (e.g., xe2x80x9cfd-tetxe2x80x9d). Various filamentous phage display systems are well known to those of skill in the art (see, e.g., , Zacher et al. (1980) Gene 9: 127-140, Smith et al.(1985) Science 228: 1315-1317 (1985); and Parmley and Smith (1988) Gene 73: 305-318).
A xe2x80x9cviral packaging signalxe2x80x9d is a nucleic acid sequence necessary and sufficient to direct incorporation of a nucleic acid into a viral capsid.
An assembly cell is a cell in which a nucleic acid can be packaged into a viral coat protein (capsid). Assembly cells may be infected with one or more different virus particles (e.g. a normal or debilitated phage and a helper phage) that individually or in combination direct packaging of a nucleic acid into a viral capsid.
The term xe2x80x9cdetectable labelxe2x80x9d refers to any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g. DynabeadsTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads. Those detectable labels that can be expressed by nucleic acids are referred to as xe2x80x9creporter genesxe2x80x9d or xe2x80x9creporter gene productsxe2x80x9d.
It will be recognized that fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like. Thus, for example, CdSexe2x80x94CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
The following abbreviations are used herein: AMP, ampicillin; c-erbB-2 ECD, extracellular do in of c-erbB-2; CDR, complementarity determining region; ELISA, enzyme linked immunosorbent a say; FACS, fluorescence activated cell sorter; FR, framework region; Glu, glucose; HBS, hepes buff red saline, 10 mM hepes, 150 mM NaCl, pH 7.4; IMAC, immobilized metal affinity chromatography; kon, association rate constant; koff, dissociation rate constant; MPBS, skimmed milk powder in PBS; PBS, skimmed milk powder in TPBS; PBS, phosphate buffered saline, 25 mM NaH2PO4, 125 NaCl, pH 7.0; PCR, polymerase chain reaction; RU, resonance units; scFv or scFv, single-chain Fv fragment; TPBS, 0.05% v/v TWEEN(copyright) 20 in PBS; SPR, surface plasmon resonance; Vk, immunoglobulin kappa light chain variable region; Vxcex, immunoglobulin lambda light chain variable region; Vl, immunoglobulin light chain variable region; VH, immunoglobulin heavy chain variable region; wt, wild type.